Figure 7. PIP₃ patch formation does not require an encircling actin ruffle, but patch size is regulated by Ras activity.

(A) The actin cytoskeleton is depolymerized by treatment with latrunculin-A and ruffles suppressed. Vegetative cells expressing an F-actin marker (Lifeact) and a PIP₃ marker (PH-CRAC) were treated for 15 min with the indicated amounts of latrunculin-A. The F-actin marker is undetectable in the cortex when treated with 5 μM latrunculin-A. (B) PIP₃ patches remain after ruffles are suppressed by depolymerisation of the actin cytoskeleton. SCAR remains associated with the patch edge under 1 μM latrunculin-A, but is lost when treated with 5 μM latrunculin-A. (C–E) Ruffles are not essential for PIP₃ patch formation. Treatment with latrunculin-A does not significantly change PIP₃ patch intensity, but leads to an increase in size and a decrease in number. (F–H) Patch boundaries are sharply defined and this does not depend on an enclosing ruffle. (F) Membrane fluorescence intensity across the edge of PIP₃ patches was measured (G) Measured intensity profiles along the edge of 36 patches of both treated and untreated cells, with each line representing a single patch. (H) Mean fluorescence intensity of the PIP₃ reporter along the patch edges of treated and untreated cells, obtained by averaging the profiles in the previous panel. (I) Macropinocytic cups in vegetative cells of wild-type cells are smaller than those of axenic strains. Shown is a maximum intensity projection of the F-actin reporter LimEΔcoil of a field of vegetative cells, recorded using lattice light sheet microscopy. (J) SCAR is still recruited to the edges of the PIP₃ patch of the small macropinocytic cups of wild-type cells. (K–L) Increased Ras activity leads to larger PIP₃ patches and macropinocytic cups. Ras activity was increased by knock-out of the RasGAP, NF1 (axeB is the gene encoding NF1). Parental DdB and knock-out cells were cultivated for 48 hr in axenic medium to maximally up-regulate macropinocytosis. (K) Confocal image of macropinocytic patches in wild-type DdB and axeB null cells; (L) Quantification of the patch size in both cell types. Loss of NF1 leads to a significant increase in macropinocytic patch size.

DOI: http://dx.doi.org/10.7554/eLife.20085.022

Figure 7—figure supplement 1. Basal PIP₃ patches (actin waves) are absent from wild-type cells.

(A–B) Basal PIP₃ patches become prevalent during early development of Ax2 cells. (A) Ax2 cells expressing a PIP₃ marker were washed free of growth medium and allowed to develop autonomously in KK2. Their basal surface was imaged using confocal microscopy and the fraction of cells with basal patches was determined at specified intervals. (B) Visualisation of basal patch abundance during development. A time-lapse image of the basal surface of developing Ax2 cells expressing a PIP₃ marker was resized to a width of one pixel and all frames were concatenated to yield a pseudo-kymograph. PIP₃ fluorescence intensity at the basal membrane is maximal around 3 hr after starvation. (C–D) Basal PIP₃ patches are caused by the specific loss of the RasGAP NF1. (C) Confocal image of the basal surface of a group of early starved wild-type NC66.2 cells and axenic Ax4 cells expressing a PIP₃ marker. Arrows point to PIP₃ patches. (D) Quantification of the number of basal PIP₃ patches of indicated strains during vegetative growth, early starvation and aggregation. Error bars indicate the standard deviation.

DOI: http://dx.doi.org/10.7554/eLife.20085.023